Distinctive urinary odors governed by the major histocompatibility

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Contributed by Edward Boyse, June 3, 1981. ABSTRACT. It has been ..... Thomas, L. (1975) in, Fourth International Convocation of Im- munology, eds. Neter, E.
Proc. NatL Acad. Sci. USA Vol. 78, No. 9, pp. 5817-5820, September 1981 Immunology

Distinctive urinary odors governed by the major histocompatibility locus of the mouse (chemosensory identification/Y maze/transfer of training)

M. YAMAGUCHI*, K. YAMAZAKI*, G. K. BEAUCHAMP*, J. BARDt, L. THOMASt, AND E. A. BOYSEt *Monell Chemical Senses Center, Philadelphia, Pennsylvania 19104; and tMemorial Sloan-Kettering Cancer Center, New York, New York 10021

Contributed by Edward Boyse, June 3, 1981

ABSTRACT It has been shown that major histocompatibility complex (MHC) types affect the mating choices of mice and that mice can be trained to distinguish arms of a Y maze scented by odors from MHC-congeneic mice. It is now shown that sensory discrimination of MHC types by trained mice in the Y maze is equally effective with urine as the source of odors. Trained mice, male and female, successfully distinguished between urines of MHC-dissimilar F2 segregants of an MHC-congeneic cross but not between urines of MHC-identical F2 segregants. In a control study with a transfer of training procedure, in which reward was withheld to eliminate any basis for new learning, the trained mice successfully distinguished between urines from panels of MHC-congeneic inbred and F2 segregant congeneic mice that had not previously been used as urine donors. Thus urine, which is a source of chemosensory signals in many species, is also a potent source of the MHC-determined odors that distinguish individual mice.

Precise recognition of the cells of one individual, by cells of another individual of the same species, is one of the attributes of the major histocompatibility complex (MHC), as the name of this group of linked genes implies (1). Until recently, knowledge of such MHC-determined communication was confined to immunologic settings. Now it is known that the recognitory functions ofthe MHC extend to the provision of a chemosensory system that enables mice to identify one another, by scent, according to their MHC genotypes. Thus, the MHC of the mouse (H-2) affects mating choices in this species (2-5), and mice can be trained to distinguish arms of a Y maze scented by pairs of MHC-congeneict mice or by MHC-congeneic F2 segregants of an MHC-congeneic cross (6). The phylogenetic implications of such a genetically controlled sensory communication system affecting behavioral and possibly other aspects of reproduction may be considerable, and it has been suggested that there may be a mechanistic and evolutionary link between chemospecific recognition by lymphocytes of the immune system and chemosensory recognition by primary sensory neurons of the olfactory system (7-9). In addition to providing further evidence of the relationship of MHC types to the sensory identification of individuals and confirming that this involves olfaction, the studies described below show that urine, which is a source of chemosensory signals in many species (10), is also a potent source of the MHCdistinctive odors that characterize individual mice.

MATERIALS AND METHODS Description and Use of the Y Maze. The Y maze is shown in Fig. 1 and has been described by Yamazaki et aL (6). Y-Maze Training. Training of the four mice B6c rB, B6 Y rB, B6d rK, and B6 9 rK (rB signifies reinforcement training for H2" and rK signifies reinforcement training for H-2k) used in studies 1-3 (Table 1) and study 4 (Table 2) by water deprivation and reward is described in detail elsewhere (6) and in the legend to Fig. 1. In short, the three consecutive phases of training required distinction of (i) juniper from cinnamon; (ii) B6 mice (H2b) from AKR mice (H-2k), whose genomic backgrounds are unrelated; and (iii) B6 mice from B6-H-2k congeneic mice (H2k haplotype donor, AKR), whose genomic backgrounds are similar except for H-2. The three training phases for the additional mice used in studies 5 and 6 (Table 3) required distinction of (i) juniper from cinnamon, (ii) urines of B6 mice from urines of AKR mice, and (iii) urines of H-2' homozygous F2 segregants of the congeneic cross (B6-H-2k X B6) (typed by cytotoxicity assay of lymph node lymphocytes) from urines of H-2k homozygous segregants of the same population. Urine Samples. Urine was obtained by gentle abdominal pressure. Usually a single mouse provided enough urine (0.2-0.3 ml) to cover the bottom of a Petri dish (3.8 cm in di*eameter) but sometimes two (rarely more) mice were needed. The urine samples were obtained from panels of individually numbered donors. As when mice were used as odor sources in previous studies (6), urine samples were assigned to the left or right odor boxes of the Y maze according to an appropriate series of random numbers (11). In each day's series of tests, up to 48 tests with each trained mouse, a given combination of two numbered male donors was never used more than once. For the testing of urine offemale mice (which evidently is more effective with larger samples), four sample-containing Petri dishes were placed in each odor box, the numbered female donors being selected in such a manner that a given combination of four donors was never used more than once in one day's trials and that the donor combinations were maximally diversified. Transfer of Training. The aim was to test for discrimination between urines of previously unused MHC-congeneic donor panels (of genotypes corresponding to prior training) without Abbreviations: MHC, major histocompatibility complex; rB and rK, reinforced (rewarded) for selecting H-2 in preference to H-2k and vice versa. * PROCEEDINGS uses this form rather than "congenic" to emphasize that the root is "gene," in analogy with our use "allogeneic," "syngeneic," etc.

The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Proc. Nad Acad. Sci. USA 78 (1981)

Immunology: Yamaguchi et al. Air

I

-~~Fan

Odor Box Petri Dish Containing Urine

FIG. 1. Y maze. Air drawn by a fan through a tube whose inlet is Fence

Gates

near the input vent supplying the laboratory is conducted through the

left and right odorboxes. Each odor box has a hinged lid to admit a Petri dish containing urine, the odor source. The air currents then pass to the left (L) and right (R) arms of the maze, which have hinged transparent lids. Each arm of the maze is fitted with a plastic tube perforated at the bottom to make one drop of water available. Each water tube is guarded by a fence that is raised only if the mouse enters the arm scented by the odor concordant with its training. Each arm of the maze is fitted with a gate that is lowered once the mouse has entered. If the choice is discordant, the fence is not raised, and the mouse is returned to the starting compartment (S). If the choice is concordant, the fence is raised to give access to the drop of water. The time interval in the starting compartment was set at 30 seconds to allow for changing the Petri dishes in the odor boxes and for replacing the drop of water (if indicated); after this, on a timed signal, all three gates are raised to commence the next trial. Left-right placing is decided by a series of random numbers suited to the sample size (11). The time taken for a trained mouse to make a choice is 2 or 3 seconds; the choice is made without pause, or after sniffing at the entrance to the arms, or sometimes with brief retracing from one arm to the other.

rewarding the trained mice and thus to eliminate any basis for new learning of hypothetical cues unrelated to MHC types (see Table 3). For this purpose, on certain days, the procedure for routine testing of the trained mice was changed so that no reward was given in every fourth test. For example, of 48 consecutive tests of a given trained mouse, 12 tests (tests 4, 8, 12, et seq.) would not include rewards. This schedule was repeated, at intervals, to accustom the trained mice to intermittent absence of reward and until the number of unrewarded tests was

adequate to demonstrate that the concordances for unrewarded tests (25% of the series) was roughly equal to the -80% concordance for the intercurrent rewarded tests (75% ofthe series). From that point on, urines for the unrewarded tests were supplied from previously unencountered donor panels (representing the same MHC distinction for which the mouse was trained). signifies the percentage of tests in which the choice in the Y maze conforms to training.

§ Concordance

Table 1. Discrimination of urines from H-2-congeneic mice: Results with the four trained mice originally studied in Y-maze trials with MHC-congeneic mice as odor sources Concordance,% 1st half 2nd half Complete Odor sources U* series n of series of series Trained mouse (urine) Study 8.567 64 70 58 1 B6cdrB, B6 rB, B66rK, B69rK Inbred & d: bb vs. kk 900 70 13.722 79 61 1178 2a B6c6rB, B69rB, B6drK, B6 rK F2 dd: bb vs. kk 8.106 64 70 58 805 2b B66rB, B69rB, B66rK, B6 rK F2 9 9: bb vs. kk F2 dc: kbvs. kk B6drB, B69rB 6.774 68 61 75 357 3 F2 d c: kb vs. bb B6drK,B69rK Urine samples from age-matched panels of individually numbered donors were as follows: study 1, 152 B6 and 103 B6-Hmales; study 2a, 57 H-2b and 61 H-2k homozygous males of the congeneic cross (B6-H-2k x B6)F2; study 2b, 23 H-2b and 16 H-2k homozygous female segregants; study 3, 74 F2 heterozygotes, 44 H-2b homozygotes and 53 H-2k homozygotes. * Standardized normal deviate:

2k

u= ( r-2 -

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where r = number of concordant responses. For each study, P